The outcome of tissue cryopreservation on the cellular, molecular and epigenetic characteristics of endometrial tissue and stromal cells.

RESEARCH QUESTION: What impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells? DESIGN: Cellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications. RESULTS: Native and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC. CONCLUSIONS: These findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.

Effects of human placenta cryopreservation on molecular characteristics of placental mesenchymal stromal cells.

Cryopreservation of placenta tissue for long-term storage provides the opportunity in the future to isolate mesenchymal stromal cells that could be used for cell therapy and regenerative medicine. Despite being widely used, the established cryopreservation protocols for freezing and thawing still raise concerns about their impact on molecular characteristics, such as epigenetic regulation. In our study, we compared the characteristics of human placental mesenchymal stromal cells (hPMSCs) isolated from fresh (native) and cryopreserved (cryo) placenta tissue. We assessed and compared the characteristics of native and cryo hPMSCs such as morphology, metabolic and differentiation potential, expression of cell surface markers, and transcriptome. No significant changes in immunophenotype and differentiation capacity between native and cryo cells were observed. Furthermore, we investigated the epigenetic changes and demonstrated that both native and cryo hPMSCs express only slight variations in the epigenetic profile, including miRNA levels, DNA methylation, and histone modifications. Nevertheless, transcriptome analysis defined the upregulation of early-senescence state-associated genes in hPMSCs after cryopreservation. We also evaluated the ability of hPMSCs to improve pregnancy outcomes in mouse models. Improved pregnancy outcomes in a mouse model confirmed that isolated placental cells both from native and cryo tissue have a positive effect on the restoration of the reproductive system. Still, the native hPMSCs possess better capacity (up to 66%) in comparison with cryo hPMSCs (up to 33%) to restore fertility in mice with premature ovarian failure. Our study demonstrates that placental tissue can be cryopreserved for long-term storage with the possibility to isolate mesenchymal stromal cells that retain characteristics suitable for therapeutic use.

Histone H4 hyperacetylation but not DNA methylation regulates the expression of decidualization-associated genes during induced human endometrial stromal cells decidualization.

The efficiency of endometrial stromal cells (ESC) decidualization is a critical player in successful embryo implantation and further pregnancy development. Epigenetic mechanisms strictly regulate massive changes that affect endometrium in each cycle, so investigating epigenetic patterns could help identify endometrial targets for infertility treatment solutions. The aim of our study was to analyze the changes in epigenetic modulators, histone modifications, and DNA methylation during induced human ESC in vitro decidualization. Decidualization markers and epigenetic factors' gene and protein expression levels were assessed during ESC cells in vitro decidualization, performing RT-qPCR and immunoblot tests. Furthermore, chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) analysis by the following qPCR were conducted to evaluate the level of H4hyperAc and 5-methylcytosine in the decidualization-associated gene promoter and exon regions accordingly. Our results revealed that ESC decidualization caused the down-regulation of HDAC2 and subunits of PRC2. We observed the increased global level of H4hyperAc and H3K27me3. We also demonstrated that H4hyperAc was specifically enriched in the decidualization-associated genes (WNT4, HAND2, STAT5A) promoter regions during ESC decidualization. In contrast, the DNA methylation level in these promoter regions was relatively low before ESC induction and did not vary through ESC decidualization. Our findings demonstrate that specific gene promoters' histone acetylation increases during the induced ESC decidualization, which indicates the importance of epigenetic regulation in endometrial decidualization.

Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.